Project 3 Summary Fanconi anemia (FA) is a recessively inherited syndrome characterized by bone marrow failure, cancer predisposition, and congenital anomalies. FA is a genetically heterogeneous disorder caused by mutations in any of 16 genes which function coordinately to promote DNA repair and prevent apoptosis in response to inflammatory cytokines. Treatment options for marrow failure in FA include androgens and hematopoietic stem cell transplant, but clinical outcomes have been limited by treatment-related toxicities. To address this challenge, this project will investigate and develop alternative medical therapies with greater efficacy and reduced side effects. The mechanism of action of therapeutic agents carries profound implications for FA patient prognosis. Agents that block adaptive stress responses to DNA damage may improve marrow failure but at the risk of promoting the survival of genetically damaged cells which may increase the risk of clonal evolution. Our hypothesis is that compounds that rescue hematopoiesis by protecting from DNA damage provide opportunities to develop safer and more effective therapies to treat marrow failure in Fanconi anemia. The prior funding period of this program project resulted in the identification of several small molecule compounds that improve growth and hematopoiesis in murine models and immortalized cell lines. The molecular mechanism(s) whereby these compounds rescue FA cells will be investigated in Project 1 (PI: Dr. Grompe) and Project 2 (PI: Dr. D'Andrea). Project 3 focuses on evaluating these compounds in primary FA bone marrow cells and FA induced pleuripotent stem cell (iPSC) models (Aim 1) to investigate their effects on hematopoiesis, DNA damage, DNA repair response, and clonal outgrowth. Compounds that rescue hematopoiesis by reducing DNA damage will be prioritized for clinical trial development in Aim 2. Potential biomarkers of DNA damage will be developed for correlative biological studies within the clinical trial. The successful completion with these aims will yield a clinical trial protocol and associated regulatory documents ready to implement by the completion of this project. These agents may be broadly applicable for the treatment of marrow failure in general.